Acute leukemia represents the deregulated production of undifferentiated hematopoietic cells that eventually suppresses normal hematopoiesis. Transcriptional regulation of lineage-affiliated genes is critical for hematopoietic development, and the loss of function mutations of myeloid-affiliated transcription factors such as CEBPalpha. has been found in acute myelogenous leukemia (AML) patients, suggesting that the inhibition of differentiation is critical for the leukemic transformation. Mice deficient for CEBPalpha exhibit differentiation block at myeloid progenitor stages but do not develop AML, suggesting that the inhibition of differentiation is not sufficient for leukemic transformation. Thus, it is reasonable to speculate that leukemic transformation could be obtained through at least two transformation processes; 1) a block in differentiation at a level of progenitors, and 2) an accumulation of immature cells at this stage due to an enhanced proliferation or a decreased rate of apoptotic cell death. We would like to analyze the physiological roles of myeloid transcription factors including CEBPalpha and PU.1 in lineage commitment, and test this hypothesis directly introducing these abnormalities into hematopoietic prot, enitors. We have developed Mxl-cre-CEBPalpha conditional knockout mice, in which the CEBPalpha gene can be completely excised in adult hematopoiesis. We have found that the elimination of CEBPalpha in adult hematopoiesis causes a complete differentiation block during transition from common myeloid progenitor (CMP) to granulocyte/monecyte progenitor (GMP) stages, resulting in the accumulation of CEBPalpha -/- CMPs. InAim 1, we will extensively study this model focusing on the biological activity of accumulated CEBP(alpha -/- CMPs. In Aim 2, we will investigate the role of PU.1 in lineage commitment by using mice harboring a reporter for endogenous PU.1 transcriptional activation. We will purify viable hematopoietic stem and progenitor subfractions with different PU.1 expression profiles, and evaluate the difference in their biological functions. In Aim 3, we will introduce oncogenic proteins such as p210-Bcr- Abl, AML1-ETO, and Bcl-2 into accumulated CEBPalpha -/- CMPs to test whether these abnormalities can cooperate with the loss of CEBPalpha for leukemic transformation. The set of these studies will be useful to understand the role of CEBPalpha and PU.1 in both normal and malignant hematopoiesis.